The isolation and identification of a microorganism that can naturally secrete a product of potential industrial production is one of, if not the most, vital steps in the process of fermentation biotechnology. The ability to secrete the protein of interest usually leads to easier downstream processing. The next critical stage is the mutagenesis of a naturally occurring strain to a hyper-producing strain. Over a number of years, scientists have developed screening strategies from which a number of exo-protein producing bacteria have been isolated. Following isolation, a large number of rounds of mutagenesis can be used to continuously select higher producing strains. However, classical strain improvement cannot be used indefinitely to further increase production levels. Therefore, a more direct method of characterization and molecular genetic manipulation is needed to achieve higher production levels.
Several patents and publications have claimed or described a lipase modulator gene (WO 94/02617; EP 331,376; Nakanishi et al. (1991) Lipases-Struct. Mech. Genet. Eng. GBF Monographs 16:263-266). However, later research has shown that the product of the gene, now called lif, is concerned with folding of the lipase rather than regulating the expression of the lipase. A review of various lipase expression systems that use the lif gene product can be found in Jaeger et al. (1994) FEMS Microbiol. Rev. 15:29-63.
Another publication discusses the sigma 54 promoter and the types of genes that have been described to be under control of this type of promoter. Morrett and Segovia (1993) J. Bacter. 175:6067-6074.
The search has continued for an expression system that can efficiently express a heterologous protein, particularly a lipase in Pseudomonas, in particular Pseudomonas alcaligenes. Pseudomonas expression of lipase is very difficult and often is at lower levels than industry would like to see.
The present invention solves the problem of low levels of expression of proteins in Pseudomonas as well as other microbial hosts.